Detection of relapse in acute lymphoblastic leukemia (ALL) is essential for proper management. However, immunophenotypic detection of relapse by flow cytometry in B-lineage ALL can be confounded by several factors, including lack of a unique immunophenotype and modulation of aberrant phenotypes after treatment. We hypothesized that flow cytometric DNA ploidy analysis may detect relapse in aneuploid ALL cases that might be missed by flow immunophenotyping. We retrospectively studied ALL cases at our institution between 1991 and 2003 (n = 114). Aneuploid populations were present at diagnosis in 32% of all patients. Sixty-five percent of all patients had "normal" leukemic immunophenotypes, defined as being similar to normal precursor B-cells, while 35% had "aberrant" immunophenotypes with myeloid or T antigen co-expression. In ALL cases that were originally aneuploid, follow-up ploidy-analysis detected relapsed disease in all cases which were also detected by flow immunophenotyping, suggesting that ploidy analysis is highly sensitive for detecting ALL relapse. However, in 5 cases in which the diagnosis of relapse could not be reliably made by flow immunophenotyping, ploidy analysis successfully detected aneuploid cells, i.e., relapse, in all five; these included 3 patients with normal and 2 with aberrant original immunophenotypes. These results suggest that it may be beneficial to perform ploidy analysis as an adjunct to flow immunophenotyping in following patients with B-lineage ALL who demonstrate aneuploidy at diagnosis.